Complete mitochondrial genomes of edible mushrooms: features, evolution, and phylogeny.

Edible mushrooms are an important food source with high nutritional and medicinal value. They are a useful source for studying phylogenetic evolution and species divergence. The exploration of the evolutionary relationships among these species conventionally involves analyzing sequence variations within their complete mitochondrial genomes, which range from 31,854 bp (Cordyceps militaris) to 197,486 bp (Grifolia frondosa). The study of the complete mitochondrial genomes of edible mushrooms has emerged as a critical field of research, providing important insights into fungal genetic makeup, evolution, and phylogenetic relationships. This review explores the mitochondrial genome structures of various edible mushroom species, highlighting their unique features and evolutionary adaptations. By analyzing these genomes, robust phylogenetic frameworks are constructed to elucidate mushrooms lineage relationships. Furthermore, the exploration of different variations of mitochondrial DNA presents novel opportunities for enhancing mushroom cultivation biotechnology and medicinal applications. The mitochondrial genomic features are essential for improving agricultural practices and ensuring food security through improved crop productivity, disease resistance, and nutritional qualities. The current knowledge about the mitochondrial genomes of edible mushrooms is summarized in this review, emphasising their significance in both scientific research and practical applications in bioinformatics and medicine.

Recent insights into glucans biosynthesis and engineering strategies in edible fungi.

Fungal α/ß-glucans have significant importance in cellular functions including cell wall structure, host-pathogen interactions and energy storage, and wide application in high-profile fields, including food, nutrition, and pharmaceuticals. Fungal species and their growth/developmental stages result in a diversity of glucan contents, structures and bioactivities. Substantial progresses have been made to elucidate the fine structures and functions, and reveal the potential molecular synthesis pathway of fungal α/ß-glucans. Herein, we review the current knowledge about the biosynthetic machineries, including: precursor UDP-glucose synthesis, initiation, elongation/termination and remodeling of α/ß-glucan chains, and molecular regulation to maximally produce glucans in edible fungi. This review would provide future perspectives to biosynthesize the targeted glucans and reveal the catalytic mechanism of enzymes associated with glucan synthesis, including: UDP-glucose pyrophosphate phosphorylases (UGP), glucan synthases, and glucanosyltransferases in edible fungi.

Mucor circinelloides: a model organism for oleaginous fungi and its potential applications in bioactive lipid production.

Microbial oils have gained massive attention because of their significant role in industrial applications. Currently plants and animals are the chief sources of medically and nutritionally important fatty acids. However, the ever-increasing global demand for polyunsaturated fatty acids (PUFAs) cannot be met by the existing sources. Therefore microbes, especially fungi, represent an important alternative source of microbial oils being investigated. Mucor circinelloides-an oleaginous filamentous fungus, came to the forefront because of its high efficiency in synthesizing and accumulating lipids, like γ-linolenic acid (GLA) in high quantity. Recently, mycelium of M. circinelloides has acquired substantial attraction towards it as it has been suggested as a convenient raw material source for the generation of biodiesel via lipid transformation. Although M. circinelloides accumulates lipids naturally, metabolic engineering is found to be important for substantial increase in their yields. Both modifications of existing pathways and re-formation of biosynthetic pathways in M. circinelloides have shown the potential to improve lipid levels. In this review, recent advances in various important metabolic aspects of M. circinelloides have been discussed. Furthermore, the potential applications of M. circinelloides in the fields of antioxidants, nutraceuticals, bioremediation, ethanol production, and carotenoids like beta carotene and astaxanthin having significant nutritional value are also deliberated.

The ß-1,3-glucan synthase gene GFGLS2 plays major roles in mycelial growth and polysaccharide synthesis in Grifola frondosa.

ß-1,3-Glucans are well-known biological and health-promoting compounds in edible fungi. Our previous results characterized a glucan synthase gene (GFGLS) of Grifola frondosa for the first time to understand its role in mycelial growth and glucan biosynthesis. In the present study, we identified and functionally reannotated another glucan synthase gene, GFGLS2, based on our previous results. GFGLS2 had a full sequence of 5944 bp including 11 introns and 12 exons and a coding information for 1713 amino acids of a lower molecular weight (195.2 kDa) protein with different conserved domain sites than GFGLS (5927 bp with also 11 introns and a coding information for 1781 aa). Three dual-promoter RNA-silencing vectors, pAN7-iGFGLS-dual, pAN7-iGFGLS2-dual, and pAN7-CiGFGLS-dual, were constructed to downregulate GFGLS, GFGLS2, and GFGLS/GFGLS2 expression by targeting their unique exon sequence or conserved functional sequences. Silencing GFGLS2 resulted in higher downregulation efficiency than silencing GFGLS. Cosilencing GFGLS and GFGLS2 had a synergistic downregulation effect, with slower mycelial growth and glucan production by G. frondosa. These findings indicated that GFGLS2 plays major roles in mycelial growth and polysaccharide synthesis and provides a reference to understand the biosynthesis pathway of mushroom polysaccharides. KEY POINTS: • The 5944-bp glucan synthase gene GFGLS2 of G. frondosa was cloned and reannotated • GFGLS2 showed identity and significant differences with the previously identified GFGLS • GFGLS2 played a major role in fermentation and glucan biosynthesis.

Improved glucose and xylose co-utilization by overexpression of xylose isomerase and/or xylulokinase genes in oleaginous fungus Mucor circinelloides.

Most of the oleaginous microorganisms cannot assimilate xylose in the presence of glucose, which is the major bottleneck in the bioconversion of lignocellulose to biodiesel. Our present study revealed that overexpression of xylose isomerase (XI) gene xylA or xylulokinase (XK) gene xks1 increased the xylose consumption by 25 to 37% and enhanced the lipid content by 8 to 28% during co-fermentation of glucose and xylose. In xylA overexpressing strain Mc-XI, the activity of XI was 1.8-fold higher and the mRNA level of xylA at 24 h and 48 h was 11- and 13-fold higher than that of the control, respectively. In xks1 overexpressing strain Mc-XK, the mRNA level of xks1 was 4- to 11-fold of that of the control strain and the highest XK activity of 950 nmol min-1 mg-1 at 72 h which was 2-fold higher than that of the control. Additionally, expression of a translational fusion of xylA and xks1 further enhanced the xylose utilization rate by 45%. Our results indicated that overexpression of xylA and/or xks1 is a promising strategy to improve the xylose and glucose co-utilization, alleviate the glucose repression, and produce lipid from lignocellulosic biomass in the oleaginous fungus M. circinelloides. KEY POINTS: • Overexpressing xylA or xks1 increased the xylose consumption and the lipid content. • The xylose isomerase activity and the xylA mRNA level were enhanced in strain Mc-XI. • Co-expression of xylA and xks1 further enhanced the xylose utilization rate by 45%.

ARTP mutation and adaptive laboratory evolution improve probiotic performance of Bacillus coagulans.

Bacillus coagulans is a thermophilic, facultative anaerobic, spore-forming Gram-positive bacterium, which is used as a probiotic in animal feed and human dietary supplements. In the present study, a bile-resistant thermophilic B. coagulans WT-03 strain was isolated and genetically identified. Atmospheric pressure room temperature plasma (ARTP)-induced mutation combined with adaptive laboratory evolution (ALE) was used to improve the probiotic performance of B. coagulans WT-03. After 15 s of ARTP mutation and 40 days of ALE culture, a mutant artp-aleBC15 was obtained and showed the improved tolerance to pH 2.5 and 0.3% bile salt with a survival rate of 22.4%. Further studies showed that the artp-aleBC15 mutant exhibited a relatively stable morphology, lower permeability, and higher hydrophobicity of cell membrane compared with the parent strain of B. coagulans. Additionally, artp-aleBC15 could maintain homeostasis with an intracellular pH of over 4.5 and had the altered contents of saturated fatty acids/unsaturated fatty acids in the cell membrane at pH 2.5. Our study proved that ARTP mutation combined with ALE is an efficient mutagenesis strategy to improve the probiotic performance of B. coagulans for potential industrial use.Key Points• A B. coagulans strain that can grow at 80 °C and 0.3% bile salt was screened.• ARTP combined with ALE effectively mutated B. coagulans WT-03.• B. coagulans artp-aleBC15 mutant showed an improved probiotic performance.• The mutant exhibited the lower permeability and altered fatty acid contents in the cell membrane.

Grifola frondosa Glycoprotein GFG-3a Arrests S phase, Alters Proteome, and Induces Apoptosis in Human Gastric Cancer Cells.

GFG-3a is a novel glycoprotein previously purified from the fermented mycelia of Grifola frondosa with novel sugar compositions and protein sequencing. The present study aims to investigate its effects on the cell cycle, differential proteins expression, and apoptosis of human gastric cancer SGC-7901 cells. Our findings revealed that GFG-3a induced the cell apoptosis and arrested cell cycle at S phase. GFG-3a treatment resulted in the differential expression of 21 proteins in SGC-7901 cells by upregulating 10 proteins including RBBP4 associated with cell cycle arrest and downregulating 11 proteins including RUVBL1, NPM, HSP90AB1, and GRP78 involved in apoptosis and stress response. qRT-PCR and Western blot analysis also suggested that GFG-3a could increase the expressions of Caspase-8/-3, p53, Bax, and Bad while decrease the expressions of Bcl2, Bcl-xl, PI3K, and Akt1. These results indicated that the stress response, p53-dependent mitochondrial-mediated, Caspase-8/-3-dependent, and PI3k/Akt pathways were involved in the GFG-3a-induced apoptosis process in SGC-7901 cells. These findings might provide a basis to prevent or treat human gastric cancer with GFG-3a and understand the tumor-inhibitory molecular mechanisms of mushroom glycoproteins.

Proteomics analysis of high lipid-producing strain Mucor circinelloides WJ11: an explanation for the mechanism of lipid accumulation at the proteomic level.

BACKGROUND: The oleaginous fungus, Mucor circinelloides, is attracting considerable interest as it produces oil rich in γ-linolenic acid. Nitrogen (N) deficiency is a common strategy to trigger the lipid accumulation in oleaginous microorganisms. Although a simple pathway from N depletion in the medium to lipid accumulation has been elucidated at the enzymatic level, global changes at protein levels upon N depletion have not been investigated. In this study, we have systematically analyzed the changes at the levels of protein expression in M. circinelloides WJ11, a high lipid-producing strain (36 %, lipid/cell dry weight), during lipid accumulation. RESULTS: Proteomic analysis demonstrated that N depletion increased the expression of glutamine synthetase, involved in ammonia assimilation, for the supply of cellular nitrogen but decreased the metabolism of amino acids. Upon N deficiency, many proteins (e.g., fructose-bisphosphate aldolase, glyceraldehyde-3-phosphate dehydrogenase, enolase, pyruvate kinase) involved in glycolytic pathway were up-regulated while proteins involved in the tricarboxylic acid cycle (e.g., isocitrate dehydrogenase, succinyl-CoA ligase, succinate dehydrogenase, fumarate hydratase) were down-regulated, indicating this activity was retarded thereby leading to a greater flux of carbon into fatty acid biosynthesis. Moreover, glucose-6-phosphate dehydrogenase, transaldolase and transketolase, which participate in the pentose phosphate pathway, were up-regulated, leading to the increased production of NADPH, the reducing power for fatty acid biosynthesis. Furthermore, protein and nucleic acid metabolism were down-regulated and some proteins involved in energy metabolism, signal transduction, molecular chaperone and redox homeostasis were up-regulated upon N depletion, which may be the cellular response to the stress produced by the onset of N deficiency. CONCLUSION: N limitation increased those expressions of the proteins involved in ammonia assimilation but decreased that involved in the biosynthesis of amino acids. Upon N deprivation, the glycolytic pathway was up-regulated, while the activity of the tricarboxylic acid cycle was retarded, thus, leading more carbon flux to fatty acid biosynthesis. Moreover, the pentose phosphate pathway was up-regulated, then this would increase the production of NADPH. Together, coordinated regulation of central carbon metabolism upon N limitation, provides more carbon flux to acetyl-CoA and NADPH for fatty acid biosynthesis.

Boosting fatty acid synthesis in Rhodococcus opacus PD630 by overexpression of autologous thioesterases.

OBJECTIVES: To explore the role of thioesterases in Rhodococcus opacus PD630 by endogenously overexpression in this bacteria for increased lipid production. RESULTS: Overexpression of four thioesterases from R. opacus PD630 in E. coli led to a 2- to 8-fold increase in C16:1 and C18:1 fatty acids while, when overexpressed in R. opacus PD630, only two recombinants had significant effect on the quantities and compositions of total fatty acid. The contents of total fatty acids (FAs) in two recombinants, pJTE2 (OPAG_00508 thioesterase) and pJTE4 (WP_012687673.1 thioesterase), were 400-460 mg/g (CDW) which is 1.5 times of wild-type strain PD630 (300-350 mg/g CDW), and 20-30 % (w/w) more than that of the control strain PDpJAM2 (330-370 mg/g CDW). The contents of 17:1 and 18:1 fatty acids increased by about 27 and 35 %, respectively, in pJTE2 and by 35 and 20 %, respectively, in pJTE4 compared with the control strain. CONCLUSIONS: The engineered strains showed improved production of lipid (as total fatty acids), and could also tailor the composition of the fatty acid profile when cultured in mineral salts medium using glucose as sole carbon source.

Lipase genes in Mucor circinelloides: identification, sub-cellular location, phylogenetic analysis and expression profiling during growth and lipid accumulation.

Lipases or triacylglycerol hydrolases are widely spread in nature and are particularly common in the microbial world. The filamentous fungus Mucor circinelloides is a potential lipase producer, as it grows well in triacylglycerol-contained culture media. So far only one lipase from M. circinelloides has been characterized, while the majority of lipases remain unknown in this fungus. In the present study, 47 potential lipase genes in M. circinelloides WJ11 and 30 potential lipase genes in M. circinelloides CBS 277.49 were identified by extensive bioinformatics analysis. An overview of these lipases is presented, including several characteristics, sub-cellular location, phylogenetic analysis and expression profiling of the lipase genes during growth and lipid accumulation. All of these proteins contained the consensus sequence for a classical lipase (GXSXG motif) and were divided into four types including α/ß-hydrolase_1, α/ß-hydrolase_3, class_3 and GDSL lipase (GDSL) based on gene annotations. Phylogenetic analyses revealed that class_3 family and α/ß-hydrolase_3 family were the conserved lipase family in M. circinelloides. Additionally, some lipases also contained a typical acyltransferase motif of H-(X) 4-D, and these lipases may play a dual role in lipid metabolism, catalyzing both lipid hydrolysis and transacylation reactions. The differential expression of all lipase genes were confirmed by quantitative real-time PCR, and the expression profiling were analyzed to predict the possible biological roles of these lipase genes in lipid metabolism in M. circinelloides. We preliminarily hypothesized that lipases may be involved in triacylglycerol degradation, phospholipid synthesis and beta-oxidation. Moreover, the results of sub-cellular localization, the presence of signal peptide and transcriptional analyses of lipase genes indicated that four lipase in WJ11 most likely belong to extracellular lipases with a signal peptide. These findings provide a platform for the selection of candidate lipase genes for further detailed functional study.

Grifola frondosa polysaccharides: A review on structure/activity, biosynthesis and engineering strategies.

Polysaccharides are the main polymers in edible fungi Grifola frondosa, playing a crucial role in the physiology and representing the healthy benefits for humans. Recent efforts have well elucidated the fine structures and biological functions of G. frondosa polysaccharides. The recently-rapid developments and increasing availability in fungal genomes also accelerated the better understanding of key genes and pathways involved in biosynthesis of G. frondosa polysaccharides. Herein, we provide a brief overview of G. frondosa polysaccharides and their activities, and comprehensively outline the complex process, genes and proteins corresponding to G. frondosa polysaccharide biosynthesis. The regulation strategies including strain improvement, process optimization and genetic engineering were also summarized for maximum production of G. frondosa polysaccharides. Some remaining unanswered questions in describing the fine synthesis machinery were also pointed out to open up new avenues for answering the structure-activity relationship and improving polysaccharide biosynthesis in G. frondosa. The review hopefully presents a reasonable full picture of activities, biosynthesis, and production regulation of polysaccharide in G. frondosa.

Enhancing microalgal biomass production in lab-scale raceway ponds through innovative computational fluid dynamics-based electrode deflectors.

The design of novel electrode deflector structures (EDSs) introduced a promising strategy for enhancing raceway ponds performance, increasing carbon fixation, and improving microalgal biomass accumulation. The computational fluid dynamics, based flow field principles, proved that the potency of arc-shaped electrode deflector structures (A-EDS) and spiral electrode deflector structures (S-EDS) were optimal. These configurations yielded superior culture effects, notably reducing dead zones by 9.1% and 11.7%, while elevating biomass increments of 14.7% and 11.5% compared to the control, respectively. In comparison to scenarios without electrostatic field application, the A-EDS group demonstrated pronounced post-stimulation growth, exhibiting an additional biomass increase of 11.2%, coupled with a remarkable 23.6% surge in CO2 fixation rate and mixing time reduction by 14.7%. A-EDS and S-EDS, combined with strategic electric field integration, provided a theoretical basis for promoting microalgal biomass production and enhancing carbon fixation in a raceway pond environment to similar production practices.

Recent Advances of Oxalate Decarboxylase: Biochemical Characteristics, Catalysis Mechanisms, and Gene Expression and Regulation.

Oxalate decarboxylase (OXDC) is a typical Mn2+/Mn3+ dependent metal enzyme and splits oxalate to formate and CO2 without any organic cofactors. Fungi and bacteria are the main organisms expressing the OXDC gene, but with a significantly different mechanism of gene expression and regulation. Many articles reported its potential applications in the clinical treatment of hyperoxaluria, low-oxalate food processing, degradation of oxalate salt deposits, oxalate acid diagnostics, biocontrol, biodemulsifier, and electrochemical oxidation. However, some questions still remain to be clarified about the role of substrate binding and/or protein environment in modulating the redox properties of enzyme-bound Mn(II)/Mn(III), the nature of dioxygen involved in the catalytic mechanism, and how OXDC acquires Mn(II) /Mn(III). This review mainly summarizes its biochemical and structure characteristics, gene expression and regulation, and catalysis mechanism. We also deep-mined oxalate decarboxylase gene data from National Center for Biotechnology Information to give some insights to explore new OXDC with diverse biochemical properties.

Biochemical and structural characterization of a glucan synthase GFGLS2 from edible fungus Grifola frondosa to synthesize ß-1, 3-glucan.

BACKGROUND: Grifola frondosa is a Basidiomycete fungus belonging to the family of Grifolaceae and the order of Polyporales. ß-Glucans are the main polymers in G. frondosa, playing a crucial role in the physiology and representing the healthy benefits for humans. The membrane-integrated ß-1, 3-glucan synthase (GLS) is responsible for glucan synthesis, cell wall assembly, differentiation and growth of the edible fungi. However, the structural/catalytic characteristics and mechanisms of ß-1, 3-glucan synthases in G. frondosa are still unknown due to their extremely complex structures with multi-transmembranes and large molecular masses. RESULTS: Herein, a ß-1, 3-glucan synthase (GFGLS2) was purified and identified from the cultured mycelia with a specific activity of 60.01 pmol min-1 µg-1 for the first time. The GFGLS2 showed a strict specificity to UDP-glucose with a Vmax value of 1.29 ± 0.04 µM min-1 at pH 7.0 and synthesized ß-1, 3-glucan with a maximum degree of polymerization (DP) of 62. Sequence Similarity Network (SSN) analysis revealed that GFGLS2 has a close relationship with others in Ganoderma sinense, Trametes coccinea, Polyporus brumalis, and Trametes pubescens. With the assistance of 3D structure modelling by AlphaFold 2, molecular docking and molecular dynamics simulations, the central hydrophilic domain (Class III) in GFGLS2 was the main active sites through binding the substrate UDP-glucose to 11 amino acid residues via hydrogen bonds, π-stacking and salt bridges. CONCLUSIONS: The biochemical, 3D structural characterization and potential catalytic mechanism of a membrane-bound ß-1, 3-glucan synthase GFGLS2 from cultured mycelia of G. frondosa were well investigated and would provide a reasonable full picture of ß-1, 3-glucan synthesis in fungi.

Magnetic/electric field intervention on oil-rich filamentous algae production in the application of acrylonitrile butadiene styrene based wastewater treatment.

Globally, the release of acrylonitrile-butadienestyrene (ABS) wastewater from numerous industries is a serious concern. Recently, oil-rich filamentous algae Tribonema sp has been grown utilizing toxic but nutrient-rich ABS effluent. Here, Tribonema sp. was cultivated under intervention of different magneto-electric combinatory fields (MCFs) (control, 0.6 V/cm, 1 h/d-1.2 V/cm, 1 h/d-0.6 V/cm, and 1 h/d-1.2 V/cm). Results showed MCF (1 h/d-0.6 V/cm) intervention increased the biomass by 9.7% (2.4 g/L) combined with high removal efficiencies (95% and 99%) of ammonium nitrogen and total phosphorus. The chemical oxygen demand (COD) removal rate increased to 82%, 6% higher than the control. Moreover, MCF of 1 h/d-0.6 V/cm significantly increased lipid and carbohydrate by 7.71% and 4.73% respectively. MCF increased premium fatty acid content such as palmitic acid (C16:0), myristic acid (C14: 0), and hexadecenoic acid (C16:1). MCF intervention also supported a diverse microbial flora, offering a favorable solution for ABS wastewater treatment.

Biomass production of carbohydrate-rich filamentous microalgae coupled with treatment and nutrients recovery from acrylonitrile butadiene styrene based wastewater: Synergistic enhancement with low carbon dioxide supply strategy.

This study attempted to remove acrylonitrile and acetophenone from simulated acrylonitrile butadiene styrene (ABS) based wastewater while recovering nitrogen and phosphorus using the carbohydrate-rich filamentous microalgae Tribonema sp.. Results showed that typical acetophenone and acrylonitrile presented significant inhibitory effect on Tribonema sp. growth and co-metabolism of CO2 improved the tolerance of Tribonema sp. to toxic pollutants. The microalgae biomass increased by 34.47% (3.16 g/L) and 58.17% (3.97 g/L) via supplementing 2% CO2 in the 100 mg/L acrylonitrile and acetophenone groups, respectively. The filamentous microalga was rich in carbohydrates and its productivity was further enhanced by 32.52% and 70.34%, respectively, in 100 mg/L acrylonitrile and acetophenone groups with 2% CO2 supplement. The synergistic CO2 supply strategy effectively enhanced the biomass production of filamentous microalgae, and moreover, improved the treatment efficiency of ABS based wastewater simulated by acetophenone or acrylonitrile addition, while at same time enhanced the recovery of nitrogen and phosphorus nutrients.

Functional Characterization and Structural Basis of the ß-1,3-Glucan Synthase CMGLS from Mushroom Cordyceps militaris.

ß-1,3-Glucan synthases play key roles in glucan synthesis, cell wall assembly, and growth of fungi. However, their multi-transmembrane domains (over 14 TMHs) and large molecular masses (over 100 kDa) significantly hamper understanding of their catalytic characteristics and mechanisms. In the present study, the 5841-bp gene CMGLS encoding the 221.7 kDa membrane-bound ß-1,3-glucan synthase CMGLS in Cordyceps militaris was cloned, identified, and structurally analyzed. CMGLS was partially purified with a specific activity of 87.72 pmol/min/µg, a purification fold of 121, and a yield of 10.16% using a product-entrapment purification method. CMGLS showed a strict specificity to UDP-glucose with a Km value of 84.28 µM at pH 7.0 and synthesized ß-1,3-glucan with a maximum degree of polymerization (DP) of 70. With the assistance of AlphaFold and molecular docking, the 3D structure of CMGLS and its binding features with substrate UDP-glucose were proposed for the first time to our knowledge. UDP-glucose potentially bound to at least 11 residues via hydrogen bonds, π-stacking ,and salt bridges, and Arg 1436 was predicted as a key residue directly interacting with the moieties of glucose, phosphate, and the ribose ring on UDP-glucose. These findings would open an avenue to recognize and understand the glucan synthesis process and catalytic mechanism of ß-1,3-glucan synthases in mushrooms.

Changes of structures and biosynthesis/hydrolysis-associated genes expression of glucans at different Volvariella volvacea maturity stages.

In the present study, effects of maturity stage on structural characteristics and biosynthesis/hydrolysis-associated genes expression of glucans from Volvariella volvacea fruit body were well investigated. Elongation and pileus expansion stages decreased total soluble carbohydrate and protein contents to 17.09 mg/g and 8.33 mg/g, and significantly accumulated the total amino acids contents to 32.37 mg/g. Yields of crude polysaccharides significantly increased to 8.12% at egg stage and decreased to 3.72% at pileus expansion stage. Purified VVP I-a and VVP I-b were proved to be α-glucans. The maturity process affected the monosaccharide compositions, decreased the molecular weights of VVP I-a and VVP I-b with decreased transcription levels of glucan biosynthesis-associated enzyme genes vvugp and vvgls and increased glucan hydrolysis-associated glucanase gene vvexg2 expression with no significant effects on backbone structures including glycosidic linkages and configurations. The findings would benefit for understanding change patterns of V. volvacea glucan structures and their biosynthesis/hydrolysis-associated genes expression at maturity stages.

The role of Rho1 gene in the cell wall integrity and polysaccharides biosynthesis of the edible mushroom Grifola frondosa.

Grifola frondosa polysaccharides, especially ß-glucans, showed the significant antitumor, hypoglycemic, and immune-stimulating activities. In the present study, a predominant regulatory subunit gfRho1p of ß-1,3-glucan synthase in G. frondosa was identified with a molecular weight of 20.79 kDa and coded by a putative 648-bp small GTPase gene gfRho1. By constructing mutants of RNA interference and over-expression gfRho1, the roles of gfRho1 in the growth, cell wall integrity and polysaccharide biosynthesis were well investigated. The results revealed that defects of gfRho1 slowed mycelial growth rate by 22% to 33%, reduced mycelial polysaccharide and exo-polysaccharide yields by 4% to 7%, increased sensitivity to cell wall stress, and down-regulated gene transcriptions related to PKC-MAPK signaling pathway in cell wall integrity. Over-expression of gfRho1 improved mycelial growth rate and polysaccharide production of G. frondosa. Our study supports that gfRho1 is an essential regulator for polysaccharide biosynthesis, cell growth, cell wall integrity and stress response in G. frondosa.

Enhanced Acid Tolerance in Lactobacillus acidophilus by Atmospheric and Room Temperature Plasma (ARTP) Coupled with Adaptive Laboratory Evolution (ALE).

The aim of this study was to improve the acid tolerance of Lactobacillus acidophilus by combining atmospheric and room temperature plasma (ARTP) mutation with adaptive laboratory evolution (ALE). To achieve a high mutation efficiency, 60 s was determined as the ideal exposure time for ARTP mutation of L. acidophilus with a survival rate of 5.91%. The ARTP-ALE mutant strain LAartp-ale2 displayed increased lactic acid stress tolerance with survival rates of 75.67% and 25.78% when cultured in pH 3.0 and 2.5, respectively, for 3 h. Physiological analysis revealed that the ARTP-ALE mutant exhibited a lower inner membrane permeability than that of the parental strain during acid stress. Furthermore, the mutant LAartp-ale2 produced more biofilm in response to lactic acid-induced acid stress and showed an increased hydrophobicity (87.2%) when compared to the parent strain (76.2%) at pH 2.5. LAartp-ale2 exhibited a higher unsaturated fatty acid (UFA) to saturated fatty acid (SFA) ratio that affected the physical state of the cell membrane for increased survival in pH 3.0 and 2.5. The mutation with ARTP coupled with ALE in the present study proved to be effective in enhancing the acid tolerance of L. acidophilus for potential industrial use.